Mono-oxygenases catalyse the selective oxidation of activated and unactivated carbon-hydrogen bonds using oxygen.sup.1, and are therefore of great interest for potential use in organic synthesis. However, progress in this area has been hampered by the difficulty in isolating sufficient quantities of the mono-oxygenase enzyme and/or the associated electron-transfer proteins. Despite the availability of amino acid sequences of more than 150 different cytochrome P-450 mono-oxygenases, to date structural date of only three are available.sup.2.3.4, and few have been successfully over-expressed in bacterial systems.sup.5.
One cytochrome P-450 mono-oxygenase, which is soluble and can be expressed in sufficient quantities, is the highly specific P-450cam from P. putida which catalyses the regio- and stereo-selective hydroxylation of camphor to 5-exo-hydroxycamphor.sup.6. The high resolution crystal structure of P-450cam has been determined.sup.2, and since the mechanism of action of this bacterial enzyme is believed to be very similar to that of its mammalian counterparts, it has been used as a framework on which structural models of mammalian enzymes are based.
The nucleotide sequence and corresponding amino acid sequence of P-450cam have been described.sup.5.7. The location of an active site of the enzyme is known and structure-function relationships have been investigated.sup.8.9. Mutants of P-450cam have been described at the 101 and 185 and 247 and 295 positions.sup.9.10.11. and at the 87 position.sup.12. A mutant in which tyrosine 96 (Y96) has been changed to phenylalanine 96 (the Y96F mutant) has been described.sup.11.13.14.15.. But in all cases the papers report effects of the mutations on the oxidation reactions of molecules which had previously been shown to be substrates for the wild-type enzyme. There is no teaching of how mutations might be used to provide biocatalysts for oxidation of different, novel substrates.